Increasing recombinant protein expression through improvements in transcription, translation, protein folding and/or secretion is a fundamental priority for optimizing yield during cell line development. The Chinese hamster ovary elongation factor 1-α (CHEF1) expression system has been used extensively to create clinical cell lines for producing recombinant proteins. The elongation factor 1-α (EF-1α) gene is highly expressed in most tissue types, and EF-1 is one of the most abundant proteins in human cells (Beck et al., Molecular Systems Biology 7: 549; 2011). CHEF1 expression vectors achieve high-level recombinant protein expression in Chinese hamster ovary (CHO) cells, as well as in non-hamster cells.
CHEF1 expression is coordinated with growth such that titer increases are driven by increased volumetric productivity. Typically, protein expression initiates early in the exponential phase of growth and drops off during stationary phase and decline. The linkage between protein expression and cell growth is consistent with the regulation of the native EF-1α gene, which is constitutively expressed to function in ribosomal protein complexes. Expression of EF-1α has been documented to increase in transformed (Sanders et al., Nucleic Acids Research 20: 5907; 1992) and mitogen-stimulated cells (Thomas and Thomas, Journal of Cell Biology 103: 2137; 1986), consistent with constitutive expression of EF-1α in actively growing cells. In addition to transcriptional control in growing cells, the growth factor insulin regulates the translation of EF-1α through the mRNA 5′ untranslated region (5′UTR) (Hammond and Bowman, Journal of Biological Chemistry 25: 17785; 1988; Proud and Denton, Biochemical Journal 328: 329; 1997). This translational control is achieved through the Tract of Polypyrimidine (TOP) sequence found in the 5′UTR (Mariottini and Amaldi, Molecular and Cellular Biology 10: 816; 1990).
CHEF1 expression systems have been shown to be capable of achieving higher levels of protein expression than vectors employing other commonly used promoters, such as the cytomegalovirus (CMV), human EF-1α, and Simian virus 40 (SV40) promoters (Running Deer and Allison, Biotechnology Progress 20: 880; 2004). The CMV promoter is one of the most widely used promoters for recombinant protein expression. For example, the glutamine synthetase (GS) system uses a murine or human CMV promoter (Kalwy, S., “Towards stronger gene expression—a promoter's tale,” 19th European Society for Animal Cell Technology (ESACT) meeting, 2005). The commercial expression plasmid pcDNA™3 (Life Technologies Corp., Carlsbad, Calif.) carries a CMV promoter derived from the major immediate-early (IE) gene (GenBank Accession #K03104.1) described previously (Boshart et al., Cell 1985; 4:521). Another commonly used CMV promoter is derived from the human CMV strain AD169 (GenBank Accession #X17403.1), also known as human herpesvirus 5.
Vectors containing CHEF1 regulatory DNA result in improved expression of recombinant proteins that is up to 280-fold greater than from CMV-controlled plasmids (Running Deer and Allison, 2004). Increased expression of a variety of proteins of interest, including secreted and membrane-bound proteins, has been achieved in different eukaryotic cell lines, including non-hamster cells, using CHEF1-driven vectors. Transfection efficiencies between CHEF1 and CMV vectors are comparable, but expression levels in clones transfected with CHEF1 vectors are generally uniformly higher.
Despite the demonstrated success of CHEF1 vectors in driving high-level expression of recombinant proteins, there exists an ongoing need to develop improved expression systems.